K. mikimotoi has been classified under many names and has been mis-assigned to different species and genera in the North Atlantic and Pacific because of its morphological similarities to other Gymnodinoid species. It is now known to be widely distributed, but there remain unresolved questions about whether K. mikimotoi was introduced into the North Sea from Japanese waters, or whether it has always inhabited this region and been erroneously classified as Gymnodinium spp. or has been a part of the hidden flora prior to be recognised in a bloom off the Norwegian coast in 1966. To address questions about geographical genetic variation within K. mikimotoi and broader issues about its biogeography it was deemed important to develop a suitable diagnostic molecular marker that could then be used to monitor the presence/absence of different K. mikimotoi ecotypes over long time scales in European waters. This study showed that the partial rDNA LSU (D1-D2) was too conserved to separate the different strains of K. mikimotoi, while, the ITS region was better able to discriminate between the different strains. However, the rbcL gene was the most informative gene and contained sufficient substitutions to separate the different strains of K. mikimotoi. Specific PCR-primers were designed to amplify a variable region of the rbcL gene able to distinguish differences between K. mikimotoi isolates from the different regions. The innovative high resolution melting temperature (HRM) technique based on specific primer set allowed rapid discrimination of K. mikimotoi from distinct geographic localities (= sequence variants) that differed by only a single nucleotide. Moreover, this study used archival environmental samples collected from the Celtic Sea shelf-break region. The high resolution melting temperature assay successfully detected the European K. mikimotoi isolate within the south-western English Channel in a 1963 sample, which is prior to thefirst report of a K. mikimotoi bloom in Norwegian waters in 1966 and in the south-western English Channel in 1975 and in western Japan in 1965. HRM observations were further validated using clone libraries and sequencing. In summary, this data provided more information about the genotypes present over the analysed timescales, revealing that K. mikimotoi sub-species 2 (European and New Zealand strains) was present in south-western English Channel and south-west Ireland for over 47 years, with sub-species 1 (the Japanese isolate) being absent from all examined samples. This finding supports the hypothesis that K. mikimotoi isolates within Europe are not of Japanese origin and suggests that they are native species to the region.

Harmful algal blooms (HABs) in the Gulf of Mexico are primarily caused by dense aggregations of the dinoflagellate species, Karenia brevis. Karenia brevis produces a highly toxic neurotoxin, brevetoxin which has been shown to cause Neurotoxic Shellfish Poisoning (NSP) and respiratory distress in humans in addition to a wide range of negative impacts upon natural ecosystems. Karenia mikimotoi is a co-existing species present during K. brevis blooms. K. mikimotoi has caused major HAB events in other parts of the ocean, but has not been recognized as a major contributor to toxicity of blooms in the Gulf of Mexico. K. brevis and K. mikimotoi have both been associated with the presence of unidentified hemolytic toxins. Production of hemolysins has not previously been investigated for either species to date in the Gulf of Mexico. Presence of hemolysins may affect toxicity and the overall impact of HABs. Therefore, detection of hemolysins is imperative for accurate identification of potential harmful impacts of such blooms. The primary goal of this research is to define whether either species is capable of producing hemolytic activity independent of brevetoxin activity; and to identify if there is significant differentiation between a variety of clonal isolates regarding toxicity and growth rate when subjected to variable experimental conditions.

Harmful algal blooms (HABs) in the Gulf of Mexico are primarily caused by dense aggregations of the dinoflagellate species, Karenia brevis. Karenia brevis produces a highly toxic neurotoxin, brevetoxin which has been shown to cause Neurotoxic Shellfish Poisoning (NSP) and respiratory distress in humans in addition to a wide range of negative impacts upon natural ecosystems. Karenia mikimotoi is a co-existing species present during K. brevis blooms. K. mikimotoi has caused major HAB events in other parts of the ocean, but has not been recognized as a major contributor to toxicity of blooms in the Gulf of Mexico. K. brevis and K. mikimotoi have both been associated with the presence of unidentified hemolytic toxins. Production of hemolysins has not previously been investigated for either species to date in the Gulf of Mexico. Presence of hemolysins may affect toxicity and the overall impact of HABs. Therefore, detection of hemolysins is imperative for accurate identification of potential harmful impacts of such blooms. The primary goal of this research is to define whether either species is capable of producing hemolytic activity independent of brevetoxin activity; and to identify if there is significant differentiation between a variety of clonal isolates regarding toxicity and growth rate when subjected to variable experimental conditions.

Harmful algae studies, in particular toxic dinoflagellates, and their response to global warming in the Red Sea are still limited. This study was aimed to be the first to characterize the identity, thermal responses and toxin production of four Prorocentrum strains isolated from the Central Red Sea, Saudi Arabia. Morphological and molecular phylogenetic analysis identified the strains as P. elegans, P. rhathymum and P. emarginatum. However, the identity of strain P. sp.6 is currently unresolved, albeit sharing close affinity with P. leve. Growth experiments showed that all species could grow at 24-32°C, but only P. sp.6 survived the 34°C treatment. The optimum temperatures (Topt) estimated from the Gaussian model corresponded to 27.17, 29.33, 26.87, and 27.64°C for P. sp.6, P. elegans, P. rhathymum and P. emarginatum, respectively. However, some discrepancy with the Topt derived from the growth performance were observed for P. elegans and P. emarginatum, as thermal responses differed from the typical Gaussian fit. The Prorocentrum species examined showed a sharp decrease after the optimum temperature resulting in very high activation energies for the fall slope, especially for P. elegans and P. emarginatum. The minimum critical temperature limit for growth was not detected within the range of temperatures examined. Subsequently, high performance liquid chromatography coupled with mass spectrometry (HPLC-MS) analysis revealed all species as non okadaic acid (OA, common toxin of the Prorocentrum genus) producers at any temperature treatment. However, other forms of toxin (i.e. fast acting toxins) not examined here could be produced. Therefore, further investigations are required. The results of this study provided significant contribution to our knowledge regarding the presence, thermal response and toxin production of four Prorocentrum species from the Central Red Sea, Saudi Arabia.

Interest in the effect of fish farming practices on the marine environment has arisen because there is concern that the wastes that fish farms produce may be contributing to eutrophication in coastal areas and the problem of harmful algal blooms. The focus of this thesis is an examination of phytoplankton distribution and abundance in relation to tuna fish farms in Boston Bay and near-shore Spencer Gulf. This is the first study in South Australia to define the short-term biomass fluctuations of chlorophyll and in vivo fluorescence, identify phytoplankton species distribution and abundance, including two potentially toxic dinoflagellates, and describe patch distribution relative to tuna fish farms in Boston Bay and the near-shore waters of Spencer Gulf. An ecological interpretation of phytoplankton distribution and abundance is determined and shows that community composition was different in lower Spencer Gulf compared to Boston Bay and upper Spencer Gulf sites. Pico- and nanophytoplankton were often the most abundant organisms. Diatoms and gymnoids were most common. Season and currents predominantly influenced the distribution of phytoplankton in Boston Bay and Spencer Gulf. Individual species may be influenced by inputs from the fish farms. Chlorophyll levels were different between the Spencer Gulf and Boston Bay sites and no differences were recorded, using mean levels of chlorophyll, between tuna cages and controls. Chlorophyll levels were higher east of Boston Island in autumn of 1999. Chlorophyll levels appeared to show a slight increase between years. This may have been an anomalous natural variation and future research may investigate this in the long term. In addition, Principal Components Analysis (PCA) was used to investigate differences between treatments and the functional grouping model supported an ecological interpretation of the factors from the PCA. A total of 131 taxa of phytoplankton were identified in this study. The 14 dominant taxa were used in the PCA and of these, 9 were diatoms. Phytoplankton abundance was not different between tuna cages and controls. However, when examining individual species, Karenia mikimotoi was more prevalent at tuna cages, close to shore, east and west of Boston Island than at other sites. PCA showed how different species bloomed together and were seasonal. Karenia brevis and K. mikimotoi featured predominantly in the PCA with K. brevis the dominant organism during summer and autumn along with Gyrodinium spp. and smaller gymnoids. K. brevis blooms were most likely influenced by water temperatures and fixation of nitrogen from a Trichodesmium erythaeum bloom. K. mikimotoi bloomed bimodally and may be influenced by ammonia excreted from fish from the tuna farms but , on the other hand, may be limited by the high salinities of South Australian waters. Currents in the region distribute both organisms. The final aspect of this study assessed finer temporal and spatial sampling using directional transects around tuna cages and controls using in-vivo fluorescence and size fractionated chlorophyll. The chlorophyll a sampling showed little spatial variability within a site in the 1000 m2 that the sampling area covers but far greater temporal variability (days). In contrast, fluorescence `mapping' expands the window of variability both spatially (within a site) and temporally (along transects and between days). This has given a spatial definition, which is unavailable from a single point sample, and thereby leaves room for much greater interpretation. Small patches are evident from the fluorescence mapping where this is impossible to detect from the single point samples. Therefore, the fluorescence `mapping' and patch definition show that the trend is widespread (spatially) and quite persistent (temporally) around the fish farm area. Size fractionated chlorophyll samples provided further insight into phytoplankton dynamics in this study where diatoms were favored over dinoflagellates and were responsible for the larger fraction of chlorophyll found at the tuna cage one (TC1) site. We suggest that seasonal fluctuations, high nutrient input from the farm activities and turbulence may be responsible for the different chlorophyll/fluorescent structures found at TC1. Future research may look at the long-term regional impact on phytoplankton size structure, biomass and communities from fish farm activities. As a good part of this journey involved counting phytoplankton using the Utërmohl technique, a short paper, published in the Journal of Plankton Research, on reducing the settling time of this method, is presented in Appendix.

No description available.

No description available.

This dissertation includes 3 chapters which focus on algal bloom of the tidal fresh James River. The first chapter describes nutrient and light limitation assays performed on algal cultures and draw conclusions about long-term patterns in nutrient limitation by comparing results with a previous study . This chapter also describes the influence of riverine discharge upon nutrient limitation in a point-source dominated estuary. This chapter was published in Estuaries and Coasts (Wood and Bukaveckas 2014). The second chapter presents the first comprehensive assessment of the occurrence of the cyanotoxin Microcystin in water and biota of the James River. Data presented in this chapter show that bivalve grazing declines in the presence of Microcystin in the water. The chapter also describes feeding habits in fish as a predictor for inter-specific differences in Microcystin accumulation in their tissues. The work presented in this chapter was published in Environmental Science & Technology (Wood et al. 2014). The third chapter describes the fate of algal carbon in the James River Estuary and the importance of autochthonous and allochthonous sources of organic matter in supporting production of higher trophic levels. Here I draw upon ecosystem metabolism data (NPP and R), abundance and grazing estimates for primary consumers and estimates of advective losses of chlorophyll and external inputs of nitrogen to place ‘top-down’ effects in the broader context of factors influencing chlorophyll and nitrogen fluxes in the James. . This chapter also describes results from mesocosm experiments used to assess the influences of grazers on chlorophyll, nutrients and Microcystis. This work will be submitted in the summer of 2014 to the journal Ecosystems.

Domoic acid (DA) is a neurotoxin produced by the harmful algae Pseudo-nitzschia that has been directly linked to mass stranding events of the California Sea Lion (CSL). The purpose of this paper is to review the anthropogenic influence on the production of this neurotoxin and examine how human activities are impacting this marine mammal species. A comprehensive and interdisciplinary literature review was conducted to evaluate the future sustainability of the CSL population. It was found that while Pseudo-nitzschia bloom developments are vulnerable to anthropogenic influences, the incontestable existence of natural contributing factors adds a certain complexity to the determination of causalities and the development of solutions. Strong evidence exists to show that DA can cause major and irreversible neurological damage in CSLs. Rehabilitation of DA-impacted CSLs is a polarizing issue in the U.S. and presents interesting implications for sustainable development. While the CSL population is currently healthy and plenteous, the strong abundance of future uncertainties warrants concern. A balance must be found between the involving social, economic, and environmental factors to ensure a promising future for the CSL species.

The marine diatom genus Pseudo-nitzschia includes species that produce domoic acid, a neurotoxin responsible for illness and mortality in both humans and marine wildlife. Because of the expertise and time required for the microscopic discrimination of species, molecular methods that monitor environmental concentrations of Pseudo-nitzschia provide a rapid alternative for the early detection of blooms and prediction of toxin accumulation. We have developed a nucleic acid sequence-based amplification with internal control RNA (IC-NASBA) assay and a quantitative reverse transcription PCR (qRT-PCR) assay for the detection of the toxic species P. multiseries targeting the ribulose- 1,5-biphosphate carboxylase/oxygenase small subunit (rbcS) gene. Both methods use RNA amplification and fluorescence-based real-time detection. Due to a limited rbcS sequence database, primers were designed and used to sequence this gene from 14 strains of Pseudo-nitzschia (including four P. multiseries) and 19 other marine diatoms. The IC-NASBA and qRT-PCR assays had a limit of detection of one cultured cell of P. multiseries and were linear over four and five orders of magnitude, respectively (r2 ! 0.98). Neither of the assays detected closely related organisms outside the Pseudo-nitzschia genus, and the qRT-PCR assay was specific to P. multiseries. While cross-reactivity of primers with unknown species prevented reliable detection of P. multiseries in spiked environmental samples using IC-NASBA, the qRT-PCR assay had positive detection from 107 cells/L to 103 cells/L. Nearly a 1:1 relationship was observed between predicted and calculated cell concentrations using qRT-PCR. Based on a diel expression study, the rbcS transcript copy number per cell ranged from 2.16 x 104 to 5.35 x 104, with the highest expression during early to mid photoperiod. The rbcS qRT-PCR assay is useful for the detection and enumeration of low concentrations of P. multiseries in the environment.